US5268360A - Opioid peptides derived from wheat proteins - Google Patents

Opioid peptides derived from wheat proteins Download PDF

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US5268360A
US5268360A US07/801,388 US80138891A US5268360A US 5268360 A US5268360 A US 5268360A US 80138891 A US80138891 A US 80138891A US 5268360 A US5268360 A US 5268360A
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fraction
peptide
tyr
seq
opioid
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Masaaki Yoshikawa
Shin-ichi Fukudome
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Nisshin Seifun Group Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/70—Enkephalins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to new peptides having opioid activity derived from wheat proteins, in particular wheat gluten, processes for the preparation thereof and pharmaceutical compositions comprising those peptides as active ingredient.
  • peptides derived from food proteins possess opioid activities such as morphine-like narcotic, analgesic activities and may be of physiological importance.
  • FIG. 1 shows the separation of opioid active fractions I, II, III and IV from an octadecyl silane (ODS) column.
  • ODS octadecyl silane
  • FIG. 2 shows the separation of opioid active fraction V from the ODS column.
  • the present invention provides peptides having opioid activity (opioid peptides) which are prepared from a hydrolysis of wheat proteins by proteases.
  • opioid peptides of the invention contain those with the determined structure and those with the undetermined structure, both of which are included within the scope of the invention.
  • those with the determined structure can include opioid peptides, each having the following amino acid sequence:
  • opioid peptides each having the following amino acid sequence
  • the amino acids constituting the opioid peptides of the invention may be in any of D-, L- and DL-configurations.
  • the peptides of the present invention can be prepared by new enzymatic process which comprises hydrolysis of wheat proteins by an acid protease, further hydrolysis of the hydrolysates by a neutral protease or an alkaline protease and optionally fractionation, isolation and purification of the resultant peptide mixture by conventional means.
  • the amino acid sequence of the peptides thus isolated may be decided for example by use of a protein sequencer.
  • the peptides of the present invention can also be synthesized by a conventional method, e.g. a solid-phase synthesis according to the t-Boc strategy.
  • the wheat proteins which are usable in the invention can include gluten alone and a mixture of gluten with other proteins in wheat such as albumin, gliadin, globulin.
  • the acid proteases which are usable in the invention can include pepsin and aspartic proteinases derived from Pycnoporus coccineus, Aspergillus and Penicillium. They may be used alone or in combination therewith so long as it is not adversely affected each other. If a plurality of acid proteases are used, hydrolysis may be performed with simultaneous use of varying proteases or successive use of each protease.
  • the neutral proteases which are usable in the invention can include metal proteases derived from microorganisms including heat resistant proteases from Bacillus such as thermolysin, those derived from Aspergillus, Streptomyces and Rhizopus; those derived from plants such as papain, bromelain; and those existing in vivo of animals such as trypsin, chymotrypsin, plasmin and thrombin.
  • metal proteases derived from microorganisms including heat resistant proteases from Bacillus such as thermolysin, those derived from Aspergillus, Streptomyces and Rhizopus; those derived from plants such as papain, bromelain; and those existing in vivo of animals such as trypsin, chymotrypsin, plasmin and thrombin.
  • the alkaline proteases which are usable in the invention can include cerin proteases derived from Bacillus or the like.
  • the neutral or alkaline protease may be used alone or in combination therewith so long as it is not adversely affected each other. If a plurality of neutral proteases are used, hydrolysis may be performed with simultaneous use of varying proteases or successive use of each protease.
  • HPLC high performance liquid chromatography
  • RPC reversed-phase chromatography
  • ODS octadecyl silane
  • the peptides of the invention possess opioid activities which are involved in analgesic, narcotic, affection, respiration, pulsation, body temperature, gastrointestinal function, athrocytosis, immunity, regulation of hormone secretion such as insulin and somatostatin, enhanced absorption of electrolyte and regulated contraction of myocardium, which may be useful as an analgesic and narcotic agent, a hypnotic agent, a secretion enhancer of gastrointestinal hormone, an absorption enhancer of electrolyte or the like.
  • opioid peptides each having the amino acid sequence of formulas (1) to (6) or physiologically acceptable salts thereof.
  • the opioid peptides or their salts of the invention because of being a water soluble white powder can be administered orally or parenterally as they are or in the form dissolved in water.
  • the opioid peptide may be formulated with a wide variety of physiologically acceptable carriers.
  • the dosage forms may include tablets, capsules, suppositories, troches, syrups, granules, powders, injectable solutions or suspensions. An appropriate dose can be chosen depending on the dosage form, age and sex of patients, degree of symptom, etc.
  • the opioid peptides of the invention can be added to foods and feeds.
  • Wheat gluten is hydrolyzed with proteases to prepare a water-soluble peptide mixture.
  • wheat gluten is hydrolyzed with an acid protease in such a situation that it is dispersed and dissolved in an acid solution such as diluted hydrochloric acid.
  • the hydrolysate is neutralized or made alkaline and further hydrolyzed with a neutral or alkaline protease to prepare a peptide mixture.
  • the peptide mixture containing Peptides A (SEQ ID NO:1), B (SEQ ID NO:2), E (SEQ ID NO:5) and F (SEQ ID NO:6) can be obtained by use of the neutral or alkaline protease.
  • the peptide mixture containing Peptide D (SEQ ID NO:4) can be obtained by using as the neutral protease those existing in vivo of animals such as trypsin, chymotrypsin or the like. In this case, trypsin and chymotrypsin may be applied simultaneously or successively.
  • pepsin is preferably used as the acid protease, since the desired product can be obtained in high yield.
  • thermolysin derived from Bacillus or those derived from Aspergillus are preferably used as the neutral proteases, since the desired product can be obtained in high yield.
  • Acid, neutral and alkaline proteases may be used in the free or immobilized state.
  • the amount of protease used may be in the range of about 5,000 to 100,000 units per 100 g of dry gluten.
  • the activity (unit) of protease used is assayed by Casein-Folin coloration A method described in S. Akabori, "Method of Studying Enzymes", vol. 2, p. 238, 1961 using as a substrate 1% Hamerstein casein solution available from American Merk Co. The reaction is conducted at 30° C. for 30 min. The amount of enzyme required for liberating 1 ⁇ g of tyrosin for one minute is expressed as 1 unit.
  • a protease treatment is advantageously performed under optimum conditions of pH, temperature, amount of protease, rate and time of treatment which are chosen according to situations such as kinds and use forms of protease, etc.
  • an aqueous solution or aqueous dispersion of gluten is adjusted to the pH between about 1.5 and 5.0 and hydrolyzed with pepsin at a temperature of about 30° to 50° C.
  • the solution is adjusted to the pH between about 6.0 and 8.0 and further hydrolyzed with thermolysin or trypsin-chymotrypsin at a temperature of about 30° to 50° C. until a solubility in 0.75 M trichloroacetic acid is in the range of about 40 to 80%.
  • proteases are deactivated by heating and/or adjustment of pH.
  • the deactivated enzyme and any insoluble solid materials such as unhydrolyzed gluten are separated and removed by suitable means such as centrifugation to recover a peptide mixture from the residual solution by drying or the like.
  • the peptide mixture dissolved in water is subjected to HPLC, e.g., HPLC on a reversed column to isolate the desired peptide in a pure form.
  • HPLC e.g., HPLC on a reversed column
  • Isolation of opioid peptides from the peptide mixture can be accomplished in the following manner.
  • peptide mixture prepared by hydrolysis of wheat gluten with pepsin and then thermolysin is subjected to HPLC on an octadecyl silane (ODS) column (e.g., Cosmosil 5 C 18 -AR, Nacalai Tesque Inc.) and eluted with an aqueous 0.05% trifluoroacetic acid (TFA) solution (solution A) and an acetonitrile (ACN) solution containing 0.05% TFA (solution B) with a linear gradient between 0 to 40% the solution B.
  • ODS octadecyl silane
  • TFA trifluoroacetic acid
  • ACN acetonitrile
  • a peak fraction (fraction I) eluting in the ACN concentration between about 22.5% and 23.5%, a peak fraction (fraction II) eluting between about 24% and 25%, a peak fraction (fraction III) eluting between about 28% and 29% and a peak fraction (fraction IV) eluting between about 34% and 35% are fractionated respectively and those fractions are determined for opioid activity.
  • step (b 1 ) The opioid active fractions prepared in step (a 1 ) are subjected to reversed-phase chromatography (RPC) on a column with different characteristics from one used in (a 1 ) (e.g., Cosmosil 5 Ph, Nacalai Tesque Inc.) and eluted in a similar manner as in step (a 1 ).
  • RPC reversed-phase chromatography
  • fraction I-1 A peak fraction (fraction I-1) eluting in the ACN concentration between about 24% and 25% is separated from fraction I
  • a peak fraction (fraction II-1) eluting between about 33% and 34% is separated from fraction II
  • a peak fraction (fraction III-1) eluting between about 30% and 31% is separated from fraction III
  • a peak fraction (fraction IV-1) eluting between about 36% and 37% is separated from fraction IV.
  • Those fractions are determined for opioid activity.
  • step (c 1 ) The opioid active fractions prepared in step (b 1 ) are subjected to reversed-phase chromatography (RPC) on a column with different characteristics from one used in (b 1 ) (e.g., Cosmosil 5 CN R, Nacalai Tesque Inc.) and eluted in a similar manner as in steps (a 1 ) and (b 1 ).
  • RPC reversed-phase chromatography
  • fraction I-2 A peak fraction (fraction I-2) eluting in the ACN concentration between about 18% and 19% is separated from fraction I-1
  • a peak fraction (fraction II 2) eluting in the ACN concentration of about 27% is separated from fraction II-1
  • a peak fraction (fraction III-2) eluting between about 26% and 27% is separated from fraction III-1
  • a peak fraction (fraction IV-2) eluting between about 32% and 33% is separated from fraction IV-1.
  • Those fractions are determined for opioid activity.
  • fractions I-2, III-2 and IV-2 are subjected to the column used in (a 1 ) and then eluted with 10 mM KH 2 PO 4 --Na 2 HPO 4 buffer (pH 7) (solution C) and 50% ACN solution containing 10 mM KH 2 PO 4 --Na 2 HPO 4 buffer (pH 7) (solution D) with a linear gradient between 0% and 100% the solution D.
  • fraction I-3 A peak fraction (fraction I-3) eluting in the ACN concentration of about 18% is separated from fraction I-2, a peak fraction (fraction III-3) eluting at about 21% is separated from fraction III-2 and a peak fraction (fraction IV-3) eluting at about 28% is separated from fraction IV-2.
  • Those fractions are determined for opioid activity.
  • fraction IV-3 is subjected to the column used in (a 1 ) and then eluted by a similar procedure as in steps (a 1 ). (b 1 ) and (c 1 ). A peak fraction (fraction IV-4) eluting in the ACN concentration of about 31% is separated from fraction IV-3 which is determined for opioid activity.
  • fraction IV-4 is subjected to reversed phase chromatography on a column with different characteristics from one used steps (a 1 ), (b 1 ) and (c 1 ) (e.g., Develosil PhA-5, Nomura Chemical Inc.) and then eluted in a similar manner as in step (d 1 ).
  • a peak fraction (fraction IV-5) eluting in the ACN concentration of about 29% is separated from fraction IV-4 which is determined for opioid activity.
  • step (h 1 ) The amino acid sequence of the dried materials obtained in step (g 1 ) is analyzed by a protein sequencer (e.g., 477 A protein sequencer, Applied Biosystems Inc.). Fraction I 3 was identified as Peptide B (SEQ ID NO:2), Gly-Tyr-Tyr-Pro-Thr, fraction II-2 as Peptide A (SEQ ID NO:1), Gly-Tyr-Tyr-Pro, fraction III-3 as Peptide F (SEQ ID NO:6), Tyr-Gly-Gly Trp and fraction IV-5 as Peptide E (SEQ ID NO:5), Tyr-Gly-Gly-Trp-Leu.
  • a protein sequencer e.g., 477 A protein sequencer, Applied Biosystems Inc.
  • step (b 2 ) The opioid active fraction (fraction V) prepared in step (a 2 ) is subjected to reversed-phase chromatography (RPC) on a column with different characteristics from one used in (a 2 ) (e.g., Cosmosil 5 Ph, Nacalai Tesque Inc.) and eluted in a similar manner as in step (a 2 ).
  • RPC reversed-phase chromatography
  • a peak fraction (fraction V-1) eluting in the ACN concentration of about 33% is separated to identify the opioid activity.
  • step (c 2 ) The opioid active fraction (fraction V-1) prepared in step (b 2 ) are subjected to reversed-phase chromatography (RPC) on a column with different characteristics from one used in steps (a 2 ) and (b 2 ) (e.g., Cosmosil 5 CN-R, Nacalai Tesque Inc.) and eluted in a similar manner as in steps (a 2 ) and (b 2 ).
  • RPC reversed-phase chromatography
  • a peak fraction (fraction V-2) eluting in the ACN concentration between about 27% and 28% is separated to identify the opioid activity.
  • step (d 2 ) The opioid active fraction (fraction V-2) prepared in step (c 2 ) is subjected to the column used in step (a 2 ) and then eluted with 10 mM KH 2 PO 4 -Na 2 HPO 4 buffer (pH 7) (solution C) and 50% ACN solution containing 10 mM KH 2 PO 4 --Na 2 HPO 4 buffer (pH 7) (solution D) with a linear gradient between 0% and 100% solution D.
  • a peak fraction (fraction V-3) eluting in the ACN concentration of about 24% is separated to identify the opioid activity.
  • step (f 2 ) The amino acid sequence of the dried materials obtained in step (e 2 ) is analyzed by a protein sequencer (e.g., 477 A protein sequencer, Applied Biosystems Inc.). Fraction V-3 was identified as Peptide D (SEQ ID NO:4).
  • a protein sequencer e.g., 477 A protein sequencer, Applied Biosystems Inc.
  • Peptides A to F (SEQ ID NOS:1-6) of the present invention can be prepared by chemical synthesis, e.g., by the following method.
  • the peptide is synthesized by a Sam 2 peptide synthesizer (Biosearch Inc.) in accordance with a standard protocall of the synthesizer.
  • a Boc(butoxycarbonyl)-Pro-resin is treated with a deblock solution containing 45% trifluoroacetic acid and then coupled with Boc-Tyr(Cl 2 -Bzl) in the presence of diisopropyl carbodiimide.
  • the resin is coupled with successive, Boc Tyr(Cl 2 Bzl) and Boc-Gly to obtain Boc-Gly-Tyr(Cl 2 -Bzl) Tyr(Cl 2 -Bzl)-Pro-resin.
  • This peptide resin is placed in hydrogen fluoride containing 10% anisol and stirred at 0° C. for 1 hr. After hydrogen fluoride is distilled away, the residue is washed with ether and a peptide is extracted with 30% acetic acid. Purification of a crude peptide by HPLC on the ODS column gives Peptide A (SEQ ID NO:1).
  • Peptides B-F (SEQ ID NOS:2-6) can be also synthesized by a similar procedure as mentioned above.
  • the fractions shown in FIG. 1 was determined for opioid activity. Opioid activity was found in fraction I eluting between about 22.5 and 23.5% in the ACN concentration, fraction II eluting between about 24 and 25%, fraction III eluting between about 28 and 29% and fraction IV eluting between about 34 and 35%. (3) The same procedure as in (2) was repeated using 2000 mg of the wheat gluten hydrolysate as prepared in (1) to obtain opioid active fractions of fractions I, II, III and IV.
  • each of eluting fractions was determined for opioid activity. Opioid activity was found for fraction 1 in fraction I 1 eluting in the ACN concentration of about 24 to 25%, for fraction II in fraction II-1 eluting in the ACN concentration of about 33 to 34%, for fraction III in fraction III-1 eluting in the ACN concentration of about 30 to 31% and for fraction IV in fraction IV eluting about 36 to 37%.
  • each of eluting fractions was collected and assayed for opioid activity.
  • Opioid activity was found for fraction I-1 in a peak fraction (fraction I-2) in the ACN concentration of about 18 to 19%, for fraction II-1 in a peak fraction (fraction II-2) in the ACN concentration of about 27%, for fraction III-1 in a peak fraction (fraction III-2) in the ACN concentration of about 26 to 27% and for fraction IV in a peak fraction (fraction IV-2) in the ACN concentration of about 32 to 33%.
  • Fractions I-2, III-2 and IV-2 of the opioid active fractions as prepared in (4) above were subjected to reversed-phase chromatography (RPC) on an octadecyl silane column (Cosmosil 5 C 18 -AR, 20 mm I.D., 250 mm length, Nacalai Tesque Inc.) and then eluted with 10 mM KH 2 PO 4 --Na 2 HPO 4 buffer (pH 7) (solution C) and 50% ACN solution containing 10 mM KH 2 PO 4 --Na 2 HPO 4 buffer (pH 7) (solution D) with a linear gradient from 0 to 100% solution D. Each eluting fraction was collected and assayed for opioid activity.
  • RPC reversed-phase chromatography
  • Opioid activity was found for fraction I-2 in a peak fraction (fraction I-3) in the ACN concentration of about 18%, for fraction III-2 in a peak fraction (fraction III-3) in the ACN concentration of about 21% and for fraction IV-2 in a peak fraction (fraction IV-3) in the ACN concentration of about 28%.
  • Fraction IV-4 of the opioid active fraction prepared in (6) was further subjected to a phenetyl column (Develosil PhA-T-5, 4.6 mm I.D., 250 mm length, Nomura Chemical Inc.) and eluted under the same conditions as in (5).
  • the determination of each eluting fraction for opioid activity indicates that opioid activity was found in a peak fraction (fraction IV-5) in the ACN concentration of about 29%.
  • the yields of Peptides A, B, E and F were determined from the molecular weights and molecular extinction coefficient at Abs. 280, indicating about 4 ⁇ 10 -2 (%), 4 ⁇ 10 -3 (%), 1 ⁇ 10 -5 (%) and 3 ⁇ 10 -4 (%), successively.
  • the Rf values of Peptides A, B, E and F were determined by thin layer chromatography on silica gel plates using butanol, acetic acid, pyridine, water (15/3/10/12) as a developing solvent, indicating 0.58, 0.54, 0.77 and 0.61, successively.
  • the solution was heated at 90° C. for 20 minutes to deactivate the enzymes.
  • the hydrolysate was centrifuged at 3500 G for 20 minutes to remove an insoluble matter and the resulting supernatant was lyophilized to yield about 3.0 g of a wheat gluten hydrolysate.
  • the fractions shown in FIG. 2 was determined for opioid activity. Opioid activity was found in fraction V eluting between about 31 and 32% in the ACN concentration.
  • the dried material as prepared above was investigated for the amino acid sequence using the same model 477-A protein sequencer as used in Example 1. It was found that L-Tyr, L-Pro, L-Ile, L-Ser and L-Leu were successively liberated from the N terminus, which was identified as Peptide D (SEQ ID NO:4) having the amino acid sequence of Tyr-Pro-Ile-Ser-Leu, all of the constituent amino acids being L-amino acids.
  • the yield of Peptide D from Wheat gluten was determined from the molecular weights and molecular extinction coefficient at Abs. 280, indicating about 4 ⁇ 10 -4 (%).
  • the Rf value of Peptide D was determined by thin layer chromatography on silica gel plates in the same manner as in Example 1, indicating 0.73.
  • Boc removed-Pro resin was then washed with methylene chloride, neutralized with methylene chloride containing 10% diisopropylethylamine and further washed with methylene chloride.
  • This resin was mixed with 5 ml of a solution of 0.4 M Boc-Tyr(Cl 2 -Bzl) in dimethylformamide and 5 ml of a solution of 0.4 M diisopropylcarbodiimide in methylene chloride and a mixture was placed into the reaction vessel and reacted at room temperature for 2 hrs. under stirring.
  • This resin was introduced into 20 ml of hydrogen fluoride containing 10% anisol and stirred at 0° C. for 2 hrs to liberate a peptide from the resin.
  • Example 1 Analysis by the same protein sequencer as used in Example 1 identified the synthesized peptide as Peptide A (SEQ ID NO:1) having the amino acid sequence of Gly-Tyr-Tyr-Pro. Rf value by thin layer chromatography (TLC) was found to be 0.58 as in the product of Example 1.
  • mice Two mouse vas deferens excised from male ICR mice weighing 30-35 g were connected to an isotonic transducer (TB 612-T, Nippon Koden Kogyo Inc., Japan) with 0.4 g tension. Each of the mouse vas deferens was immersed in a 2 ml magnus tube maintained at 36° C. in which Krebs solution was filled comprising 118 mM NaCl, 4.75 mM KCl, 2.54 mM CaCl 2 , 25 mM NaHCO 3 , 1.19 mM KH 2 PO 4 and 11 mM glucose and ventilation (95% O 2 , 5% CO 2 ) was performed from bomb.
  • Krebs solution was filled comprising 118 mM NaCl, 4.75 mM KCl, 2.54 mM CaCl 2 , 25 mM NaHCO 3 , 1.19 mM KH 2 PO 4 and 11 mM glucose and ventilation (95% O 2 , 5% CO 2 ) was performed from bomb

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US5798337A (en) * 1994-11-16 1998-08-25 Genentech, Inc. Low molecular weight peptidomimetic growth hormone secretagogues
US5925731A (en) * 1995-09-26 1999-07-20 Takeda Chemical Industries, Ltd. Endothelin antagonist peptides
US20030139348A1 (en) * 1994-11-16 2003-07-24 Genentech, Inc. Low molecular weight peptidomimetic growth hormone secretagogues
US6828415B2 (en) * 1993-02-19 2004-12-07 Zentaris Gmbh Oligopeptide lyophilisate, their preparation and use
US20080058264A1 (en) * 2006-08-30 2008-03-06 Ajinomoto Co. Inc Amino-acid containing composition for inhibiting accumulation of fat
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US6828415B2 (en) * 1993-02-19 2004-12-07 Zentaris Gmbh Oligopeptide lyophilisate, their preparation and use
US6863891B2 (en) 1993-02-19 2005-03-08 Zentaris Ag Oligopeptide lyophilisate, their preparation and use
US6867191B2 (en) 1993-02-19 2005-03-15 Zentaris Gmbh Preparation and use of oligopeptide lyophilisate for gonad protection
US20050124546A1 (en) * 1993-02-19 2005-06-09 Jurgen Engel Oligopeptide lyophilisate, their preparation and use
US7605121B2 (en) 1993-02-19 2009-10-20 Aeterna Zentaris Gmbh Oligopeptide lyophilisate, their preparation and use
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US6034216A (en) * 1994-11-16 2000-03-07 Genentech Inc. Low molecular weight peptidomimetic growth hormone secretagogues
US20030139348A1 (en) * 1994-11-16 2003-07-24 Genentech, Inc. Low molecular weight peptidomimetic growth hormone secretagogues
US7094869B2 (en) 1994-11-16 2006-08-22 Genentech, Inc. Low molecular weight peptidomimetic growth hormone secretagogues
US5925731A (en) * 1995-09-26 1999-07-20 Takeda Chemical Industries, Ltd. Endothelin antagonist peptides
US20080058264A1 (en) * 2006-08-30 2008-03-06 Ajinomoto Co. Inc Amino-acid containing composition for inhibiting accumulation of fat
US9192593B2 (en) * 2006-08-30 2015-11-24 Ajinomoto Co., Inc. Amino-acid containing composition for inhibiting accumulation of fat
WO2009043455A3 (en) * 2007-09-11 2009-09-03 Mondobiotech Laboratories Ag Therapeutic uses of angiogenin 108-122 and gluten exorphin a5
WO2009043466A3 (en) * 2007-09-11 2009-09-03 Mondobiotech Laboratories Ag Therapeutic use of the peptide gluten exorphin a5
WO2009043466A2 (en) * 2007-09-11 2009-04-09 Mondobiotech Laboratories Ag Therapeutic use of the peptide gluten exorphin a5
WO2009043455A2 (en) * 2007-09-11 2009-04-09 Mondobiotech Laboratories Ag Therapeutic uses of angiogenin 108-122 and gluten exorphin a5
US20110200574A1 (en) * 2010-02-02 2011-08-18 Jolly James F Use of proteases for gluten intolerance
EP2531521A1 (de) * 2010-02-02 2012-12-12 Amano Enzyme USA., Ltd. Verwendung von proteasen für glutenintoleranz
EP2531521A4 (de) * 2010-02-02 2014-04-02 Amano Enzyme Usa Ltd Verwendung von proteasen für glutenintoleranz
US9011842B2 (en) 2010-02-02 2015-04-21 Amano Enzyme Inc. Use of proteases for gluten intolerance
US9498520B2 (en) 2010-02-02 2016-11-22 Amano Enzyme Inc. Use of proteases for gluten intolerance
WO2011112099A1 (en) 2010-03-08 2011-09-15 Marine Bioproducts As Peptide material, and preparations and uses thereof
WO2016026017A1 (pt) * 2014-08-18 2016-02-25 Embrapa - Empresa Brasileira De Pesquisa Agropecuária Peptídeo opioide
CN105385732A (zh) * 2015-11-16 2016-03-09 中国农业科学院农产品加工研究所 一种双酶复合酶解高效制备谷朊粉短肽的方法
CN105385732B (zh) * 2015-11-16 2018-11-06 中国农业科学院农产品加工研究所 一种双酶复合酶解制备谷朊粉短肽的方法

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JPH0586094A (ja) 1993-04-06
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JP3130080B2 (ja) 2001-01-31

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